The present invention relates to a method of selecting stem cells and uses thereof.
Stem cells have the unique property of being able to reconstitute populations of cells in the body. Typically, stem cells are divided into two main groups: adult stem cells and embryonic stem cells. The importance of technologies associated with expansion of stem cells, both of adult and/or embryonic derivation is illustrated by the numerous preclinical and clinical uses of these cells in treatment of a wide range of diseases.
Unlike all current treatments relying upon surgical intervention or drugs that modulate physiological activities, stem cells provide a replacement for dysfunctional or degenerating tissue. Using stem cells, replacement therapy could dramatically change the prognosis of many currently untreatable diseases, restore function of damaged organs and correct inborn disorders of metabolism and deficiencies.
The recent discoveries that adult stem cells derived from the bone marrow can give rise to non-hematopoietic tissues suggest that these cells may have greater differentiation potential than was previously assumed and open new frontiers for their therapeutic applications [Petersen, B. E. et al. Science 1999; 284:1168-1170; Brazelton, T. R. et al. Science 2000; 290:1775-1779; Krause, D. S. et al. Cell 2001; 105, 369-377].
Studies have shown that cord blood-derived stem cells are capable of repairing neurological damage caused by brain injuries and strokes [Lu D et al. Cell Transplant. 2002; 11:275-81] and are also capable of functional and morphological incorporation into animal heart tissue [Orlic, D. et al., Proc. Natl. Acad. Sci. USA 2001; 98:10344-9].
One of the earliest clinical uses of stem cells was for performing bone marrow transplants in patients with hematological malignancies in which hematopoietic stem cells derived from the donor bone marrow were administered into the recipient subsequent to providing the recipient with a sufficient dose of radiation and/or chemotherapy. This treatment ablates not only the malignant cells but also non-malignant cells. Endogenous hematopoietic cells rarely survive myeloablative radiation, and the stroma is severely damaged. In the aftermath of ablative injury, donor hematopoietic stem and progenitor cells (HSPC) find their way to the host bone marrow where they seed and engraft to reconstitute the immune-hematopoietic system. The prevalent sources of hematopoietic stem cells and progenitors include the bone marrow, umbilical cord blood and cells mobilized to the peripheral blood.
In addition to treatment of hematological malignancies, stem, progenitor and immune cells have been utilized in the context of therapy for solid tumors. Thus, for example, the use of autologous hematopoietic cell transplants combined with high dose chemo/radiotherapy for solid tumors has been extensively investigated for breast [Peppercorn, et al., 2005, Cancer 104:1580-1589]; colon [Leff, et al., J Clin Oncol 1986; 4:1586-1591], lung [Ziske, et al., Anticancer Res 2002; 22:3723-3726], nasopharyngeal cancer [Chen, et al., Jpn J Clin Oncol 2003; 33:331-335], and other types of cancers [Gratwohl, et al., Ann Oncol 2004; 15:653-660].
The identification of the type 1 transmembrane protein/adhesion molecule, the sialomucin CD34 as a marker of hematopoietic stem cells led to the use of CD34+ cell selection as a means of concentrating hematopoietic stem cell activity [Civin, et al., J Immunol 1984; 133:157-165]. Specifically, it was demonstrated that although bone marrow mononuclear cells contain approximately 1-4% CD34+ cells, the administration of these cells, but not bone marrow depleted of CD34+ cells, into lethally irradiated baboons led to hematopoietic reconstitution [Berenson, et al., J Clin Invest 1988; 81:951-955]. Similarly, CD133 has been considered as a marker of hematopoietic stem and progenitor cells [Kobari L, et al., J Hematother Stem Cell Res. 2001; 10:273-281]. Notably, the prevalence of stem cells in the bone marrow is much lower, in the order of 0.2-0.5%.
The above described method of isolating CD34+ or CD133+ cells results in a mixed cell population of stem and progenitor cells that includes all lineages and stages of lympho-hematopoietic stem and progenitor cells and some later precursor cells. This is disadvantageous, since it has been shown to be beneficial to isolate only the most primitive of the cells within the CD34+ cell population [Askenasy N. et al., Current Stem Cell Research and Therapy 2006; 1:85-94]. Such positive selection procedures additionally suffer from some disadvantages including the presence of materials such as antibodies and/or magnetic beads on the CD34+ cells, and damage to the cells resulting from the removal of these materials.
Furthermore, recent evidence suggests that expression of CD34 on the cell membrane does not always correlate with stem cell activity. It has been shown that in humans, there is a highly quiescent population of stem cells that lacks CD34 expression, but has full reconstituting capacity [Dao et al., Leukemia 2000; 14:773-776]. Hematopoietic progenitors have been repeatedly shown to be limited in their pluripotent differentiation potential, as compared to adult bone marrow and umbilical cord blood-derived stem cells that lack these phenotypic markers [Jang Y Y, et al., Nat Cell Biol. 2004; 6:532-539].
Accordingly, there is a continued interest in finding other methods to either replace or augment current methods of isolating cell populations that are enriched in stem cells and primitive progenitor cells.
Stem and progenitor cells are often required to perform differentiation tasks under extreme conditions of injury and inflammation. In this process, the expression and activation of death receptors in the developing hematopoietic cells have been attributed various functional roles, in particular negative regulation of differentiated cells, however the involvement of the death receptors in the proximal stages of HSPC function is unclear. The mechanisms by which hematopoietic reconstituting cells flourish in such devastated environment is of particular interest, as it may be used to improve the efficiency of engraftment.
There are more than 40 distinct ligand-receptor systems that are currently recognized as belonging to the tumor necrosis factor (TNF) superfamily. The majority of TNF ligands, most prominent Fas-ligand (FasL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), are synthesized as membrane-bound proteins and soluble forms are released by proteolysis. Various cell types store FasL in vesicles, which are excreted upon activation by various physiological stimuli. Within minutes from expression, FasL is cleaved from the cell surface by matrix metalloproteinases and accumulates as a soluble molecule. The soluble and membranous forms differ in their function with respect to apoptosis and immune regulation. Apoptosis is primarily mediated by the membrane-bound FasL, while the biology of the soluble isoform of FasL (sFasL) is complex, and includes apoptotic, anti-apoptotic, and chemotactic activities. Antiapoptotic (s)FasL competes with the membranous form for Fas binding, and is a chemotactic factor for neutrophils.
Expression of the Fas receptor in hematopoietic stem and progenitor cells (HSPC) is variable and changes along the lineage differentiation. Subpopulations of immature CD34+ CD38− human cells derived from the fetal liver, umbilical cord blood (UCB) and adult bone marrow (BM) were shown to express low (but detectable) levels of this receptors and other apoptosis mediating TNF receptors [Niho et al, Curr Opin Hematol. 1998; 5:163-165].
During hematopoietic cell differentiation, the Fas receptor is expressed in proliferating and differentiating progenitors, serving as a negative regulator of distal differentiation in all lineages [Gaur U, Aggarwal B B, Biochem Pharmacol. 2003; 66:1403-1408; Greil R, et al., Crit Rev Immunol. 2003; 23:301-322]. Such enhanced expression of Fas has been observed in cultured hematopoietic progenitors, and was associated with impaired viability and reduced clonogenesis following ex vivo cell exposure to cytokines, expansion and manipulation.
The TNF superfamily receptors and ligands have until presently been considered to be involved in increasing HSPC sensitivity to apoptotic signals under various experimental conditions. It was assumed that the excessive expression of Fas in HSPC exposed to injury signals following transplantation promotes the execution of apoptosis in donor cells and is involved in suppression of donor cell activity. Ex vivo incubation of human CD34+ HSPC and murine c-ki+lin−SCA-1+ (KLS) HSPC with TNF-α was associated with increased expression of the Fas receptor and resulted in deficient homing and engraftment [Bryder D, J Exp Med 2001; 194: 941-952; Dybedal I, Blood. 2003; 102:118-126]. All these detrimental effects were efficiently induced by activating anti-Fas antibodies and were reversed by blocking anti-Fas antibodies and soluble Fas-ligand. In corroboration with the negative role attributed to the Fas receptor in engraftment of HSPC, marrow hypoplasia caused by graft versus host disease was ameliorated by injection of FasL-defective cells [Iwasaki T, et al., Cell Immunol. 1999; 197:30-38].
Civin et al [U.S. Appl. No. 20040131599] teach a method for suppressing the immune response of a recipient mammal to a donor hematopoietic stem cell graft by expressing a recombinant FasL gene in donor hematopoietic stem cells. The positive role of FasL in this context was attributed to the killing of reactive T lymphocytes of the host to ameliorate allorejection, and of the donor to ameliorate graft versus host disease.
Shirwan et al [U.S. Appl. No. 20040018170] teach a method for treating conditions which are alleviated by the apoptosis of activated lymphocytes. Specifically Shirwan et al disclose the use of proteins, for example stable tetramers of FasL, in order to enhance the efficiency of activation of death receptors in activated 5 lymphocytes.
Both methods use ligands for death receptors to eliminate reactive immune cells through activation-induced cell death [Cohen J J, Duke R C. Ann Rev Immunol. 1992; 10:267-293].
However, neither Civin et al, nor Shirwan et al suggest nor allude to selection of stem cells or purification of stem cell populations prior to transplant.
Josefsen et al., [Exp Hematol. 1999; 27:1451-1459] teach promotion of CD34+ CD38− cell viability and enhancement of cytokine induced clonogenicity by addition of soluble FasL. Similar results were obtained with CD34++CD38− fetal liver cells [Barcena et al., Exp Hematol. 1999,27:1428-1439]. In this case, soluble FasL was used to inhibit apoptosis mediated by activation (trimerization) of the Fas receptor [Askenasy N, et al., Blood. 2005; 105:1396-404], which is expressed in a significant fraction of human cells transplanted in human subjects [Saheki K, et al., Br J Haematol. 2000; 109:447-452] and in murine models of xenotransplantation [Dybedal I, et al., Blood. 2003; 102:118-126]. However, the use of Fas-L as an agent to select for stem cells was not suggested.